The continuing goal of HD25256 is to determine how the unique characteristics of the newborn intestinal circulation contribute to the pathogenesis of necrotizing enterocolitis, the most common acquired GI disease of the newborn. In the past funding cycle, we determined that mechanical reduction of flow into the intestine of newborn subjects (i.e., 3- days) caused a progressive increase in gut vascular resistance that occurred over the course of hours. The early vasoconstriction (5-30 minutes) was absent when endogenous NO synthesis was blocked; as well, this rise could be attenuated by blockade of endothelin ETA receptors. The late vasoconstriction could exhibit vasoconstriction under these circumstances. We hypothesize that basal vascular tone in newborn intestine is determined by a dynamic balance between constitutively active dilator and constrictor force, whereas tone in older intestines is determined mainly by static, architectural phenomenon. We hypothesize that the low resting tone in newborn intestine reflects abundant, flow-mediated endothelium-derived NO production. We further contend that constitutively constrictor tone is also present, mediated by ETl; the physiologic effect of ET1 is not appreciated, however, for three reasons: i) the system is near maximal dilation, ii) NO decreases the binding affinity of ETA receptors, and iii) developmentally regulated ETB receptors produce a offsetting vasodilation via NO production We hypothesize that flow reduction rapidly changes this situation: NO production decreases and ETA binding affinity increases early, and AT1 expression increases hours later. This rapid shift in balance acts as an amplification system, so that otherwise minor perturbations in localized gut flow spread and worsen. To test this hypothesis, the project has four specific aims: 1) demonstrate that the mechanostimulus of wall shear stress is tightly linked to NO production in 3-, but not 35-day old intestine by directly measuring NO in the effluent of buffer-perfused mesenteric arcades and by determining the relationship between flow, shear stress and vascular diameter in mesenteric arterioles, 2) demonstrate that constitutive dilator and constrictor forces set basal vascular tone in 3-, but not 35-day old intestine by selectively blocking endogenous NO synthesis, ETA and ETB receptors under a variety of hemodynamic conditions in mesenteric arterioles and blood perfused gut loops, and also by determining the ontogeny of ETA and ETB receptors in endothelial and vascular smooth muscle cells using the TaqMan system, 3) demonstrate that the ETA receptor binding affinity is contingent upon coupling to G proteins and that NO interferes with this coupling by exposing cultured vascular smooth muscle cells to an exogenous NO donor and then measuring radioligand binding kinetics and KTPase activity, and 4) demonstrate that up-regulation of AT1 receptors and ACE activity occur in vascular smooth muscle from mesenteric arteries exposed in vivo to sustained low flow perfusion by determining expression of AT1 and ACE activity occur in vascular smooth muscle from mesenteric arteries exposed in vivo to sustained low flow perfusion by determining expression of AT1 and ACE activity in endothelial and vascular smooth muscle cells. These data should provide justification for moving the next renewal cycle of this work into studying receptor biochemistry in tissue removed from human infants undergoing bowel resection of NEC or non-NEC reasons.